Role of N6-methyladenosine in tumor neovascularization

Tumor neovascularization is essential for the growth, invasion, and metastasis of tumors. Recent studies have highlighted the significant role of N6-methyladenosine (m6A) modification in regulating these processes. This review explores the mechanisms by which m6A influences tumor neovascularization, focusing on its impact on angiogenesis and vasculogenic mimicry (VM). We discuss the roles of m6A writers, erasers, and readers in modulating the stability and translation of angiogenic factors like vascular endothelial growth factor (VEGF), and their involvement in key signaling pathways such as PI3K/AKT, MAPK, and Hippo. Additionally, we outline the role of m6A in vascular-immune crosstalk. Finally, we discuss the current development of m6A inhibitors and their potential applications, along with the contribution of m6A to anti-angiogenic therapy resistance. Highlighting the therapeutic potential of targeting m6A regulators, this review provides novel insights into anti-angiogenic strategies and underscores the need for further research to fully exploit m6A modulation in cancer treatment. By understanding the intricate role of m6A in tumor neovascularization, we can develop more effective therapeutic approaches to inhibit tumor growth and overcome treatment resistance. Targeting m6A offers a novel approach to interfere with the tumor’s ability to manipulate its microenvironment, enhancing the efficacy of existing treatments and providing new avenues for combating cancer progression.


INTRODUCTION
Tumor neovascularization ensures the acquisition of adequate oxygen and nutrients required for sustained tumor growth [1].
Notably, solid tumors tend to grow around blood vessels and cannot expand beyond 2 mm 3 without vascularization [2,3].The induction of the "angiogenic switch", which depends on a balance of angiogenic and anti-angiogenic factors, is a ratelimiting step in tumorigenesis, triggering exponential tumor growth [4,5].Neovascularization, considered as a hallmark of cancer, is indispensable for tumor proliferation, invasion, and metastasis [5].Consequently, targeting tumor neovascularization has emerged as a crucial component of cancer therapy.Existing anti-angiogenic strategies primarily focus on the vascular endothelial growth factor (VEGF) or VEGF receptor (VEGFR) signaling pathway.Despite advancements, these approaches yield transitory benefits and often fail to achieve long-term clinical responses [6].Increasing evidence indicates that tumor neovascularization is a complex process involving multiple components, underscoring the need to elucidate the underlying mechanisms to improve anti-angiogenic therapy efficacy [7][8][9].
Recently, researchers have proposed that non-mutational epigenetic reprogramming facilitates the acquisition of hallmark capabilities by tumors [10,11].Epigenetics refers to the study of heritable alterations that do not involve changes to the DNA sequence, including DNA and RNA methylation, nucleosome remodeling, and histone modifications [11,12].Over 170 RNA modifications have been identified, including N6-methyladenosine (m 6 A), 5-methylcytosine (m 5 C), N7-methylguanosine, and N1methyladenosine [13].The most prevalent RNA modification among these is m 6 A, first described in 1974, which occurs as an RNA methylation at the sixth nitrogen atom of adenosine [14].m 6 A modification is a dynamic and reversible process that is installed by methyltransferases ("writers"), removed by demethylases ("erasers"), and recognized by RNA-binding proteins ("readers") [15].m 6 A participates in multiple aspects of RNA metabolism processes, including splicing, translation, stability, degradation, and nuclear export.It plays an essential role in reshaping the tumor microenvironment (TME), regulating cancer metabolism, and facilitating carcinogenesis [12,[15][16][17].
Recent evidence highlights the role of m 6 A in regulating tumor neovascularization.Our previous review linked m 6 A to immune reprogramming [16].In this review, we aim to explore the regulatory role of m 6 A in tumor neovascularization, offering a comprehensive grasp of its significance in cancer therapy.This review introduces the diverse role of m 6 A in multiple modes of neovascularization and associated signaling pathways.Additionally, we concisely outline its contribution to vascular-immune crosstalk.Finally, we discuss the current development of m 6 A inhibitors and their potential clinical applications.This review clarifies the underlying mechanism of tumor neovascularization and provides novel insights into targeting m 6 A in anti-angiogenic therapy.

TUMOR NEOVASCULARIZATION
In 1971, Folkman proposed that solid tumor growth is always accompanied by the formation of new blood vessels, suggesting that inhibiting tumor vascularization could suppress tumor growth [2].Since then, serveral modes of tumor neovascularization have been identified, including sprouting angiogenesis, vasculogenesis, intussusceptive angiogenesis, vasculogenic mimicry (VM), vessel co-option, and cancer stem cell (CSC)-derived vasculogenesis (Fig. 1) [7,8].The first three modes occur in both normal tissues and tumors, whereas the latter three are specific to tumor neovascularization.Among them, angiogenesis and VM are the most extensively studied.

Angiogenesis
Angiogenesis is the traditional process by which new blood vessels form from preexisting vessels through sprouting.Initially, endothelial cells (ECs) loosen their junctions, increasing permeability and releasing plasma proteins.Subsequently, ECs sprout, with tip cells penetrating the basement membrane, and an imbalance between matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) leads to extracellular matrix (ECM) degradation.Finally, ECs proliferate and migrate, accompanied by pericyte recruitment, resulting in the formation of new blood vessels [7,18].

Vasculogenic mimicry (VM)
The classical theory of tumor angiogenesis proposes that blood vessels are generated through ECs sprouting.However, in 1999, a paradigm shift occurred with the introduction of the concept of VM in a study focused on melanoma [19].Unlike traditional angiogenesis, VM is independent of ECs which forms vessel-like structures by tumor cells [8].Subsequent studies revealed that VM occurs not only in melanoma but also in glioma, hepatocellular carcinoma (HCC), and prostate cancer [20][21][22].Mechanistically, VM involves the epithelial-mesenchymal transition (EMT) process and differentiation of CSCs [23,24].During VM, epithelial cell markers like E-cadherin are downregulated, whereas mesenchymal cell markers like VE-cadherin, vimentin are upregulated [24,25].
In various tumor types, METTL3 typically functions as an oncogenic factor, including acute myeloid leukemia (AML), lung cancer, HCC, among others, while exhibiting an anti-oncogenic role in endometrial cancer [32].Additionally, METTL3 is implicated in therapy resistance across cancers.For instance, it promotes chemoresistance in HCC, breast cancer, and colorectal cancer (CRC), and reduces the effectiveness of immunotherapy in CRC and melanoma [33].However, despite METTL14 lacking catalytic activity and functions as an allosteric activator of METTL3, its impact on tumors may not consistently align with METTL3.METTL14 exerts an oncogenic effect in HCC and AML, but suppresses tumor progression in CRC and renal cell carcinoma (RCC) [32,34].Additionally, decreased METTL14 has been reported to promote radiotherapy resistance in esophageal squamous cell carcinoma (ESCC) and sorafenib resistance in HCC [33].This discrepancy could arise from the distinct target sites methylated by each protein within the writer complex.m 6 A methylation is preferentially enriched at DRACH (D = A, G, or U; R = G or A; H = A, C or U) motifs, suggesting numerous potential methylation sites exist within transcripts.However, the writer complex exhibits siteand transcript-specific selectivity, which may be influenced by transcription factors or histone marks that recruit the writer complex to specific genomic loci [15,35].
The reversible nature of m 6 A modification is regulated by m 6 A erasers, which are limited in specific tissues and are contextdependent [35].m 6 A demethylation is catalyzed by two enzymes, fat mass and obesity-associated (FTO) and AlkB homolog 5 (ALKBH5), both part of the ALKB enzyme family [36].FTO is the first identified m 6 A eraser that is associated with obesity and energy homeostasis [37].Initially, FTO was identified as a nucleus m 6 A demethylase, but later studies suggested its primary target might be N6,2′-O-dimethyladenosine (m 6 A m ) rather than m 6 A [37]. m 6 A typically occurs at internal sites within mRNA, whereas m 6 A m is located near the 5′cap structure.Subsequent studies indicated that FTO regulates m 6 A m modification of snRNA, thereby affecting the alternative splicing of mRNA [38].Despite a lower response rate, FTO can demethylate m 6 A and exhibit an oncogenic role.In AML, FTO is abnormally localized in the cytoplasm and exerts oncogenic effects by altering m 6 A demethylation [39].ALKBH5, another m 6 A demethylase, is highly expressed in the testis, lungs, and germ cells, but weakly in cardiac and cerebral tissues [40,41].Unlike FTO, ALKBH5 has no activity toward m 6 A m and can be induced under hypoxia conditions in tumors, promoting selfrenewal of glioblastoma and breast CSCs [42].m 6 A readers m 6 A readers are RNA-binding proteins that specifically recognize and bind to m 6 A-modified RNA molecules.The YTH domaincontaining proteins, including YTH domain-containing 1-2 (YTHDC1-2) and YTH domain family 1-3 (YTHDF1-3), were the first identified m 6 A readers.YTHDC1 promotes RNA splicing and nuclear export, and YTHDC2 weakly binds m 6 A and promotes mRNA translation and degradation.The YTHDF proteins have three highly similar paralogues: YTHDF1 enhances mRNA translation, YTHDF2 promotes mRNA degradation, and YTHDF3 performs both functions [43].Additionally, m 6 A can indirectly recruit RNAbinding proteins by remodeling RNA structure, a phenomenon known as the"m 6 A-switch" [15].The insulin-like growth factor 2 mRNA binding protein (IGF2BP) family and heterogeneous nuclear ribonucleoprotein (HNRNP) family belong to this category.IGF2BP1-3 promote mRNA stability, HNRNPC/G facilitate RNA splicing, and HNRNPA2B1 promotes both RNA splicing and degradation [15].M 6 A AND TUMOR NEOVASCULARIZATION m 6 A and angiogenesis Among m 6 A writers, METTL3 and METTL14 are primarily investigated for their roles in tumor angiogenesis (Table 1).Firstly, they facilitate the expression of angiogenic factors in various cancers.For example, METTL3 directly promotes the expression of hypoxia-inducible factor 1-alpha (HIF-1α), VEGFA and tyrosine kinase (TEK) in bladder cancer (BLCA) [44,45].Similarly, METTL14 induces the expression of basic leucine zipper ATF-like transcription factor 2 (BATF2), which indirectly upregulates VEGFA secretion and promotes angiogenesis in tongue squamous cell carcinoma (TSCC) [46].Beyond VEGFA, METTL3 also regulates ECM components to modulate angiogenesis [47].In HCC and prostate cancer, METTL3 stimulates angiogenesis by increasing the expression of MMP2 and MMP9 [48][49][50].Moreover, in CRC, METTL3 enhances plasminogen activator, urokinase (PLAU) expression, which activates angiogenic factors stored in the ECM, thereby facilitating angiogenesis [51,52].Additionally, METTL3 regulates cell cycle-associated proteins.In gastric cancer (GC) and in head and neck squamous cell carcinoma (HNSCC), METTL3 upregulates centromere protein F (CENPF), ensuring an adequate blood supply for rapidly dividing tumor cells [53,54].METTL3 and METTL14 also affect metabolism and inflammation, indirectly regulating angiogenesis.In GC, METTL3 increases hepatoma-derived growth factor (HDGF) expression, promoting glycolysis, which subsequently contributes to angiogenesis and liver metastasis [55].In RCC, METTL14 activates TNF receptorassociated factor 1 (TRAF1), and indirectly facilitates angiogenesis [56].
The regulation of tumor angiogenesis by m 6 A erasers varies across different cancer types.In multiple myeloma (MM), ALKBH5 promotes angiogenesis by elevating salvador family WW domaincontaining protein 1 (SAV1) expression and activating the Hippo pathway [57].Conversely, in BLCA, ALKBH5 suppresses angiogenesis and hematogenous metastasis by inhibiting lncBLACAT3 expression, which inactivates the NF-κB pathway [58].In RCC, FTO promotes angiogenesis by inhibiting von Hippel-Lindau tumor Table 1.Tumor neovascularization regulated by m 6   A methylation in different types of cancer.suppressor (VHL) expression, whereas in intrahepatic cholangiocarcinoma (ICC), FTO suppresses angiogenesis by inducing TEA domain transcription factor 2 (TEAD2) expression [59,60].These findings demonstrate that the effects of m 6 A erasers on angiogenesis are specific to the cancer type.

Cancer type
Correlations between tumor angiogenesis and m 6 A readers have been observed in both the IGF2BP and YTH domaincontaining proteins, which exhibit opposing functions.The IGF2BP family exerts a pro-angiogenic effect by enhancing the stability of downstream genes.For instance, in lung cancer, IGF2BP2 increases the stability of fms-related tyrosine kinase 4 (FLT4; also known as VEGFR3) or thymidine kinase 1 (TK1) mRNA, leading to tumor angiogenesis and aggressiveness [61,62].Similarly, in CRC, IGF2BP2 and IGF2BP3 stabilize cyclin D1 and VEGF mRNA, thereby promoting angiogenesis [63,64].In contrast, the YTH domain-containing proteins have been demonstrated to suppress tumor angiogenesis.In lung cancer, YTHDC2 enhances the translation efficiency of lncZNRD1-AS1, further suppressing angiogenesis and tumorigenesis through the miR-942/Tensin 1 axis [65].Additionally, YTHDF2 inhibits angiogenesis in clear cell RCC (cRCC) and HCC by facilitating the degradation of target genes.In cRCC, YTHDF2 inhibits angiogenesis by increasing circPOLR2A degradation [66].In HCC, it increases the degradation of IL11 and serpin family E member 2 (SERPINE2) and thus contributing to vascular normalization [67].
However, the effect of METTL3 on VM in glioblastomas (GBM) appears to be different.One study found that METTL3 enhances the stability of BUD13 homolog (BUD13), promoting the translation of cyclin-dependent kinase 12 (CDK12) and muscleblind-like splicing regulator 1 (MBNL1).This cascade results in the upregulation of MMP2 and laminin subunit gamma 2 (LAMC2), promoting VM [71].Conversely, another study indicates that reduced METTL3 facilitates VM and is correlated with higher histopathological grade and lower overall survival [72].The contradictory results may be attributed to differences in cell line selection and sample size.Further research is needed to clarify the specific role of m 6 A on VM in different tumors.m 6 A and stemness-associated factors m 6 A and Oct4.Octamer-binding transcription factor 4 (Oct4), a member of the POU transcription factor family, is essential for stemness maintenance and differentiation of CSCs [73].Recent studies have also linked it to angiogenesis [74].Our previous research demonstrated that Oct4 regulates the differentiation of liver CSCs into tumor ECs [75].YTHDF2 interacts with the 5′UTR of Oct4 mRNA to increase its expression, thus maintaining stemness and promoting lung metastasis in HCC [76].ALKBH5 is positively correlated with Oct4 in MM and non-small cell lung cancer.Suppression of ALKBH5 reduces Oct4 expression and inhibits CSC characteristics, suggesting that ALKBH5 may induce neovascularization through CSC-derived vasculogenesis manner in these tumors [57,77].m 6 A and Sox2.SRY-box transcription factor 2 (Sox2) is a transcription factor that is essential for the self-renewal and pluripotency of stem cells.It has been demonstrated that Sox2 is capable of promoting tumor neovascularization.In ESCC, Sox2 promotes angiogenesis by inducing suprabasin expression [78].Furthermore, it contributes to VM in CRC [79].On the other hand, tumor neovascularization increases Sox2 expression, which in turn helps to maintain the CSC phenotype.In skin tumors, CSCs are found in proximity to ECs and reside within a perivascular microenvironment [80].Besides, in retinoblastoma, VEGF has been found to stimulate Sox2 expression and enhance tumor invasiveness [81].These findings demonstrate a reciprocal relationship between Sox2 and tumor neovascularization, where both factors reinforce the aggressive behavior of the tumor.
M 6 A AND TUMOR NEOVASCULARIZATION-ASSOCIATED PATHWAYS m 6 A and VEGF VEGF, an extensively studied angiogenic factor, is produced by various cell types.The VEGF family consists of six members, with VEGFA being the most critical for tumor neovascularization.VEGFR are categorized into three subtypes, with VEGFR1 and VEGFR2 being the most prevalent in vascular ECs.VEGFR1 is responsible for hematopoiesis, and VEGFR2 is involved in vasculogenesis and angiogenesis.VEGFR3, mainly expressed in lymphatic ECs, is associated with lymphangiogenesis [86].When VEGF binds to VEGFR, it triggers TEK phosphorylation, activating intracellular signaling pathways that regulate the proliferation, migration, survival, and penetration of vascular ECs, ultimately leading to neovascularization [18].Moreover, VEGF infulences tumor neovascularization thorugh various downstream signaling pathways, including PI3K/AKT, MAPK, PLC, and SRC [87].
In lung cancer, METTL3 binds to the A859 site within the internal ribosome entry site of VEGFA 5'UTR, recruiting YTHDC2/ eIF4GI complex to promote VEGFA translation and increase its expression [88].This promoting effect of METTL3 on VEGFA is also observed in CRC, pancreatic cancer and BLCA [45,69,89].However, in sorafenib-resistant HCC, METTL3 exerts an opposite effect.Depletion of METTL3 increases the expression of VEGFA and other angiogenic factors [90].This may result from the alterations in the recognition site of the m 6 A-modified target gene following drug resistance, which affecting their binding capacity.In RCC, FTO is mutually exclusive with VHL.Elevated FTO inhibits VHL expression and increases VEGFA secretion [59].IGF2BP3 has been reported to promote angiogenesis by interacting with VEGFA in CRC and GC [63,69,91].Notably, it is IGF2BP3, rather than other IGF2BP members, that specifically binds to VEGFA [69].Further research is required to elucidate the selectivity of m 6 A readers.
m 6 A and EGFR Epidermal growth factor receptor (EGFR) is a membrane receptor on the surface of epidermal cells that belongs to the tyrosine kinase receptor family.Upon activation by EGF, it initiates tyrosine kinase activity and activates downstream signaling such as PI3K/AKT, MAPK, and JAK/STAT pathways, which regulate various biological processes [92].
In breast cancer with brain metastases, YTHDF3 enhances the translation of EGFR and VEGFA mRNA by binding to eukaryotic translation initiation factor 3 subunit A (eIF3a).Suppressing YTHDF3 reduces blood vessel density and impairs brain endothelial tube formation, thereby decreasing brain metastasis and prolonging survival [93].Under hypoxic conditions, YTHDF2 is downregulated in HCC.However, when overexpressed, it binds to the 3'UTR of the EGFR mRNA and accelerates its degradation.Consequently, this process suppresses the MAPK/ERK pathway, thereby inhibiting cell proliferation and tumor growth [94].m 6 A and PI3K/AKT The PI3K/AKT signaling pathway is crutial in cancer development and progression.PI3K consists of a regulatory subunit (p85) and a catalytic subunit (p110).When activated, PI3K converts PIP2 to PIP3, subsequently activating PDK1 and AKT, while PTEN can counteract these effects.Upon activation, AKT stimulates mTOR, which in turn phosphorylates downstream substrates and regulates diverse biological processes [95].The PI3K/AKT pathway is widely involved in tumor neovascularization.For instance, the inactivation of the p110 subunit impedes functional vessel formation, thereby restraining tumor growth.Activated AKT in tumor ECs results in an increase in nitric oxide levels, forstering vascular permebility.Conversely, the deletion of PTEN delays pericyte maturation, resulting in defective vascular remodeling [96][97][98].
m 6 A and Hippo The Hippo pathway, comprising FZD2, LATS1/2, SAV1, and YAP/ TAZ, is essential for regulating CSC maintenance, angiogenesis, and drug resistance.FZD2 has been reported to induce VM and maintain stemness in HCC, while YAP is associated with angiogenesis and VM in various cancers [103,104].Recent studies indicate that the Hippo pathway is involved in m 6 A-mediated tumor neovascularization, especially in VM.
YAP exhibits widespread association with VM and can be mediated by m 6 A methylation.In HCC, METTL3 promotes YAP mRNA splicing and enhances its translation efficiency, thereby facilitating VM [68].In pancreatic cancer, inhibition of YTHDF2 increases YAP expression and promotes EMT [105].Using verteporfin, a YAP inhibitor, neovascularization is suppressed, as evidenced by reduced levels of angiopoietin-2 (Ang2), MMP2 and VE-cadherin [106].Given that EMT serves as the mechanism underlying VM, it is speculated that YTHDF2 may inhibit VM in pancreatic cancer by suppressing the Hippo pathway.Similarly, in CRC, IGF2BP2 binds to YAP mRNA to promote its translation, and verteporfin reduces the number of cancer-associated fibroblasts, inhibiting angiogenesis and tumor progression [107][108][109].Apart form YAP, SAV1 has been implicated in promoting stem cell phenotype and neovascularization in MM.Inhibition of ALKBH5 decreases SAV1 mRNA stability, suppresses the Hippo pathway and angiogenesis [57].Considering the intimate connection among CSC, EMT and VM, it is worthwhile to investigate the role of m 6 A in regulating tumor neovascularization, especially in VM through the Hippo pathway.
m 6 A and Wnt/β-catenin The Wnt signaling pathway is important for regulating CSC maintenance and tumor metastasis, with three distinct pathways indentified: Wnt/β-catenin, Wnt/planar cell polarity, and Wnt/ calcium.In the classical Wnt/β-catenin pathway, Wnt binds to the Frizzled receptor and activates the TCF/LEF transcription factor.Subsequently, TCF/LEF binds to β-catenin and promotes the transcription of various downstream genes [110].

M 6 A IN TUMOR VASCULAR-IMMUNE CROSSTALK
Tumor angiogenesis creates an immunosuppressive microenvironment, aiding tumors in evading immune surveillance.Additionally, immunosuppressive cells can stimulate blood vessel formation, establishing abberrant communication between vascular and immune cells [112].The synergistic effect of combining anti-angiogenic therapy with immunotherapy has demonstrated significant efficacy in treating various cancers, including RCC, HCC, GC, and endometrial cancer [113][114][115][116]. Recent studies highlight the role of m 6 A in regulating both tumor neovascularization and the immune microenvironment [117].m 6 A-mediated tumor vasculature effects on immune cells Immune cells extravasation into the TME requires adherence to ECs.However, ECs create an immune barrier by expressing programmed cell death-1 ligand 1 (PD-L1) and FAS ligand (FASL), as well as inhibiting adhesion factors such as intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and P-selectin (Fig. 3) [118,119].Additionally, angiogenic factors like VEGF impede dendritic cells (DCs) maturation, impairing antigen presentation, suppressing tumor-specific cytotoxic T lymphocytes (CTLs) activation, or promoting the accumulation of immunosuppressive cells such as myeloidderived suppressor cells (MDSCs) and regulatory T cells (Tregs) [120].METTL3 and IGF2BP3 promote VEGF expression in various cancers, potentially contributing to the immunosuppressive TME [44,63,69,88,91].
Tumor blood vessels exhibit chaotic, leaky, and highly permeable characteristics, leading to increased interstitial fluid pressure that hampers immune cell infiltration.Inhomogeneous blood flow reduces perfusion and oxygenation, adversely impacting the delivery of anticancer drugs [121].Pericytes are crucial for   maintaining blood vessel structural integrity and have dual effects on tumor neovascularization.Generally, pericytes produce angiogenic factors that stimulate neovascularization [122,123].However, under certain conditions, increased pericyte coverage can normalize tumor vasculature [5].In HCC, low levels of YTHDF2 due to hypoxia result in decreased pericyte coverage.Elevated YTHDF2 facilitates the degradation of IL11 and SERPINE2, reducing vascular density and permeability, thereby favorably impacting vascular normalization [67].
m 6 A-mediated immune cell effects on tumor neovascularization Immune cells regulate tumor angiogensis through the secretion of cytokines and chemokines, while m 6 A modification indirectly modulates this process by recruiting and activating immune cells.

M 6 A METHYLATION IN CANCER THERAPY Clinical treatment prospects of new strategies
The advancement of targeted therapy strategies focused on m 6 A regulators is rapidly progressing, driven by their crucial regulatory functions and precise recognition abilities.Dysregulation of enzymes and binding proteins involved in RNA methylation has been linked to various human cancers, suggesting a promising novel approach for clinical intervention [34].

Clinical trials and preclinical research
Ongoing clinical trials of m 6 A emphasize the potential of targeting m 6 A modifications in cancer treatment.STC-15, as an oral small molecule inhibitor targeting METTL3, is a first-in-class inhibitor of RNA modification.In November 2022, it advanced into Phase I clinical trials, representing the first RNA methyltransferase inhibitor to enter clinical development.STC-15 has demonstrated efficacy in suppressing AML or suppress tumor growth through anticancer immune responses, promising a novel avenue for cancer therapy [135].
Another promising candidate is STM2457, a highly selective METTL3 inhibitor with minimal effects on other methyltransferases, indicating its potential as a targeted cancer therapy.Preclinical research indicates that STM2457 specifically inhibits key stem cell populations in AML without significant toxicity to normal haematopoiesis, promoting cell differentiation, inducing apoptosis, and inhibiting tumor growth [136].These findings support targeting m 6 A modification as a promising strategy for anticancer therapy.

Potential of post-transcriptional modifications as biomarkers
Post-transcriptional modifications (PTMs) have been demonstrated to be associated with various human diseases.RNA modifications can be potential biomarkers for monitoring cancer progression through regulating mRNA stability, translation efficiency, and other RNA metabolic processes.Distinct modification patterns in different cancer types influence tumor cell behaviors such as proliferation, apoptosis, invasiveness, and metabolic activity, which makes them important indicators in cancer research.For instance, the m 5 C-based signature is an independent prognostic factor associated with immunotherapy efficacy and drug susceptibility in RCC [137].The interaction network of m 6 A/ m 5 C/m 1 A regulated genes is reported to assess the prognosis of HCC [138].These imply the potential of RNA modification in clinical applications.Clinically, detecting m 6 A often requires high-throughput sequencing (such as MeRIP-seq) and specific immunoprecipitation techniques [15].These methods help clinicians analyze m 6 A levels in samples to assess tumor status and treatment response.As biomarkers, m 6 A modifications have the advantage of dynamically reflecting biological changes within tumor cells, allowing real-time monitoring of tumor progression and treatment response through non-invasive samples (such as blood) [139].For instance, in gastrointestinal cancer, m 6 A levels were elevated compared to adjacent tissues and the serum of healthy individuals, and decreased post-surgery [140].However, the dynamic and variable nature of m 6 A and other RNA modifications can complicate result interpretation.Moreover, the need for specialized techniques and equipment limits the widespread clinical application of m 6 A as a biomarker.Overall, research on RNA modifications as potential cancer biomarkers is still in its early stages, requiring further study to overcome current technical barriers and enhance their clinical accuracy and feasibility.

CONCLUSIONS AND PERSPECTIVES
This review emphasizes the significant role of m 6 A modifications in tumor neovascularization.We aim to explore how m 6 A influences various modes of neovascularization and its interactions with multiple signaling pathways and components of the TME.By inhibiting key m 6 A writers or erasers, it may be possible to suppress pro-angiogenic factors, thereby reducing tumor vascularization and growth.This approach could potentially enhance the efficacy of existing treatments, such as VEGF inhibitors.
Although studies using m 6 A inhibitors to directly target tumor neovascularization are limited, growing evidence suggests that m 6 A is widely involved in anti-angiogenic resistance.Specifically, in HCC and RCC, both of which are characterized by high vascular density.Increased m 6 A levels contribute to resistance against sorafenib, apatinib, and lenvatinib in HCC.Under normoxic conditions, METTL3 increases resistance to sorafenib and lenvatinib through the Wnt/β-catenin pathway, while WTAP facilitates lenvatinib resistance under hypoxic conditions.Knockdown of METTL14 restores sensitivity to sorafenib by upregulating hepatocyte nuclear factor 3 gamma (HNF3γ).In RCC, downregulation of METTL14 enhances sensitivity to sunitinib by reducing TRAF1 expression.YTHDC1 increases sensitivity to sunitinib by inhibiting histone deacetylase 2 (HDAC2).Conversely, in pazopanib-resistant cRCC, YTHDF2 fails to recognize lncIGFL2AS1 for degradation, promoting drug resistance.These findings suggest targeting m 6 A might provide a novel approach to anti-angiogenic therapy.In conclusion, the regulatory role of m 6 A in tumor neovascularization is critical and warrants further investigation to develop potential treatment strategies.Additional research is needed to fully understand its clinical potential, especially its interactions with various pathways and immune cells.Despite the limited studies targeting tumor neovascularization with m 6 A inhibitors, growing evidence suggests that this approach may offer promising therapeutic potential.

Fig. 1
Fig. 1 Modes of tumor neovascularization.There are several modes of tumor neovascularization.a Angiogenesis: blood vessels form from preexisting vessels through sprouting; b vasculogenesis: endothelial progenitor cells derived from the bone marrow are recruited and differentiate into endothelial cells to form blood vessels; c intussusception: transcapillary tissue pillars insert into the lumen of existing vessels, undergo vascular splitting, and eventually fuse to remodel the vascular network; d vascular mimicry: tumor cells form vessel-like structures; e vessel co-option: tumor cells hijack the existing vasculature and migrate along the vessel surface or infiltrate non-malignant tissues between vessels; f CSC differentiate into ECs or PCs: cancer stem cell differentiate into endothelial cells or pericytes.CSCs cancer stem cells, ECs endothelial cells, EPCs endothelial progenitor cells, PCs pericytes.

Fig. 3
Fig. 3 m 6 A regulators in vascular-immune crosstalk.Tumor neovascularization contributes to the establishment of an immunosuppressive tumor microenvironment, while immunosuppressive cells facilitate tumor angiogenesis through the secretion of pro-angiogenic factors.Immune effector cells like CD8 + CTL, M1 TAM, mDC contribute to the establishment of an anti-angiogenic TME, whereas immunosuppressive cells, such as M2-like TAM, MDSC, and iDC, promote angiogenesis.Meanwhile, VEGF reduces the expression of endothelial adhesion molecules (ICAM1, P-selectin, E-selectin) expression, inhibits DC maturation and CTL activation, increases the abundance of MDSC, consequently impedes the infiltration of immune cells.Furthermore, reduced pericyte coverage hinders blood vessel integrity and immune infiltration.m 6 A regulators play a role in modulating immune-vascular crosstalk by influencing various components such as immune cells, vascular-associated cells, angiogenic factors, and endothelial adhesion molecules.CTL cytotoxic T lymphocyte, FASL FAS ligand, iDC immature DC, M1 TAM M1like tumor-associated macrophage, M2 TAM, M2-like tumor-associated macrophage, mDC mature DC, MDSC myeloid-derived suppressor cell, PD-L1 programmed cell death-1 ligand 1, ICAM1 intercellular adhesion molecule 1, VEGF vascular endothelial growth factor.

Table 2 .
Fundamental functions of m6A regulators and their effect on tumor neovascularization.